Viral-like particles are structures specialized in packaging and carrying nucleic acids and proteins. A general characteristic of viral-like particles is their excellent ability to stimulate the host's immune response. These properties make viral-like particles extremely interesting agents for developing intracell delivery systems and for generating sub-unit vaccines. The use of different gene expression systems has aided in producing empty viral capsids or viral-like particles (VLPs) of several viruses, for example rotavirus (US 2003/0175301), retrovirus (U.S. Pat. No. 6,602,705), parvovirus (U.S. Pat. No. 6,458,362), etc. The genetic manipulation of these expression systems in turn allows producing VLPs containing heterologous amino acid sequences from proteins different from those forming the native viral capsid. These VLPs are generically called heterotypical, recombinant or chimeric VLPs (CVLPs), and they have mainly been used for two purposes: (i) generating multivalent vaccines by means of immunogenetically relevant heterologous peptides, and (ii) modifying tropism, by means of inserting amino acid sequences involved in receptor-ligand interactions.
The infectious bursal disease virus (IBDV), which belongs to the Birnaviridae family, infects several avian species and is directly responsible for infectious bursitis, a severe immunosuppressive disease causing considerable economic losses in the avian industry worldwide.
IBDV particles are icosahedral with symmetry T=13, they lack the envelope and are formed by a single protein layer. Until now, the approaches aimed at obtaining an atomic model for IBDV particles have failed. For this reason, the available information on their structure is based on three-dimensional models generated from images obtained by electron cryomicroscopy of the purified virus and VLPs. Based on these studies, it has been observed that the outer surface of the particle is formed by a continuous lattice of 260 trimers of protein VP2 (37 kDa) arranged in five different conformations. The inner side of the particles contains 200 trimers of protein VP3 (29 kDa), the latter ones, independently of one another, are bound to the basal area of the VP2 trimers. It has been suggested that a third polypeptide, VP4 (28 kDa), could also be part of the particles, being located at the base of the pentamers forming the angles of the icosahedral structure.
Polypeptides VP2, VP3 and VP4 are produced from proteolytic processing of a precursor polypeptide with a size of 109 kDa. This precursor is auto-catalytically processed, releasing the polypeptides pVP2 (48 kDa), VP3 and VP4. The VP4 domain, which is located in the central region of the polyprotein, belongs to the family of Ion proteases and is responsible for the proteolytic cut. Polypeptides pVP2 and VP3 are directly responsible for assembling the capsids. The pVP2 product suffers a last cut at its C-terminal end before giving rise to the mature form of the protein, VP2, which is the form found in the purified particles. This pVP2 processing is necessary for correctly forming the capsids and it requires the presence of VP3, although the protease responsible has not yet been identified.
Morphogenesis is a vital process for the viral cycle requiring successive steps associated to modifications in precursor polypeptides. As a result, viruses have developed strategies allowing the sequential and correct interaction between each one of their components. One of these strategies, frequently used by icosahedral viruses, consists of the use of polypeptides of a single polyprotein as the basis of their structural components. In these cases, correct proteolytic processing of said polyprotein plays a crucial role in the assembly process.
This principle for IBDV capsid assembly has been demonstrated in earlier works (Fernández-Arias A et al. 1998. Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles. Journal of General Virology 79:1047-1054). Expression in eukaryotic cells of the gene encoding for the IBDV polyprotein gives rise to the formation of VLPs that are completely indistinguishable, both morphologically and biochemically, from IBDV virions. It has also been observed that capsid assembly requires only the synthesis and correct processing of the viral polyprotein and is independent of the presence of the viral genome or of other proteins encoded by the viral genome, such as VP5 and VP1 proteins.
Until now, results obtained from the expression of IBDV genes in different recombinant systems has allowed concluding that: i) the assembly process is independent of the presence of genetic material of the virus, ii) only those polypeptides encoded by the polyprotein gene are required for assembly, and iii) assembly requires a coordinated interaction between polypeptides VP2 and VP3.
However, it is unknown if pVP2/VP3 interaction is established between VP2 and VP3 domains of the precursor polyprotein even when it has not been modified, or if, on the contrary, this interaction occurs after processing the precursor. Furthermore, current information does not exclude the possibility that VP4 could play a relevant role in capsid morphogenesis. In fact, IBDV VLPs formed by assembly of IBDV VP2, VP3 and VP4 proteins (U.S. Pat. Nos. 6,528,063; 5,788,970 and JP 5194597) have been disclosed.
The work developed by the inventors of this work has allowed establishing systems for obtaining IBDV VLPs by using different eukaryotic expression vectors. These vectors have been used for expressing the IBDV polyprotein in the absence or presence of viral polymerase RNA VP1. The biochemical characterization of purified VLPs shows that they contain pVP2, VP2 and VP3 proteins when only the viral polyprotein is expressed, and pVP2, VP2, VP3 and VP1 proteins when the polyprotein and the viral polymerase RNA are expressed simultaneously (Fernández-Arias A et al. 1998. Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles. Journal of General Virology 79:1047-1054; Martínez-Torrecuadrada J L et al. 2000. Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells. Virology 278:322-331; Maraver A et al. 2003. The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus, plays a critical role for capsid formation. Journal of Virology 77:6438-49; Lombardo E et al. 1999. VP1, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms complexes with the capsid protein VP3, leading to efficient encapsidation into virus-like particles. Journal of Virology 73:6973-6983).
Incidentally, patent document WO 02/088339 discloses IBDV viral-like particles formed by the assembly of chimeric proteins comprising the IBDV polyprotein bound to a polypeptide at its terminal carboxyl end.
CVLPs based solely on the IBDV pVP2 protein, or on fragments thereof, fused to a polypeptide of interest, or their potential use as vaccines or as carriers for products of interest, have not been described before.